For over two thousand years Yersinia pestis, the causative agent of bubonic and pneumonic plague, has caused widespread loss of human life during recurrent pandemics. Modern, more contained epidemics are common in South America and Madagascar. In addition, Y. pestis is a category A bioterrorism agent with natural zoonotic foci as readily available sources of the organism on nearly every continent. The ability of pathogens to acquire iron from their hosts is one critical parameter in the outcome of the infectious process. Y. pestis encodes nine potential inorganic iron transport systems and two heme/hemoprotein transport systems. Of these, the siderophore-dependent yersiniabactin (Ybt) iron transport system and the Yfe ABC transporter are the most important systems for acquisition of inorganic iron. Ybt is essential in the early stages of bubonic plague and mutations in this system are avirulent in mice infected subcutaneously (SC). However, Ybt- mutants are fully virulent via an intravenous (IV) route of infection. Yfe plays an important role during the later stages of the infection - a Ybt-Yfe- mutant is completely avirulent in mice by IV injection. By SC injection, a Yfe- mutant is 75-fold less virulent than its Yfe+ parent suggesting that the Ybt system can partially compensate for a lack of the Yfe system but it is clearly not as effective in the later stages of disease. Expression of both systems is repressed by iron through the iron-responsive regulatory protein Fur. In addition, the Ybt system is activated by an AraC-type regulator, YbtA, possibly acting in concert with the Ybt siderophore. The Yfe system is also repressed by excess manganese through the Fur protein. In vivo repression by manganese and Fur is a unique regulatory mechanism. The specific aims of this proposal are to continue characterizing genetic and biochemical aspects of 1) the Ybt and 2) the Yfe transport systems and 3) to analyze the expression of these two systems in vivo and determine their roles in systemic spread of the disease. We will identify any remaining elements necessary for the function of these systems, examine the regulatory components controlling expression of these systems in vitro and in vivo, and determine the role of these systems in the infectious disease process of plague. An understanding of the components and functions of these two-transport system may lead to their use as protective antigens or as targets for new drugs. Our studies will also provide insights into the role of Ybt and Yfe in the pathogenesis of plague and other disease-causing organisms and into the general importance of iron acquisition in bacterial disease processes.